Heme A is an obligatory cofactor in eukaryotic cytochrome c oxidase (CcO), but little is known about how heme A is inserted into CcO, or how the flux of heme through the heme a biosynthetic pathway is coordinated to CcO assembly. Because CcO is indirectly responsible for ~50% of the ATP formed during aerobic metabolism, it is crucial that cells maintain a proper level of heme A, and deficiencies in heme a homeostasis lead to several clinical and fatal early-onset mitochondrial disorders. At the same time, however, excess heme a is toxic, and it is therefore critical for cells to regulate its production to ensure that there is sufficient, but not excess, heme A available. Our lack of understanding about the processes that affect heme a biosynthesis and its insertion into CcO represents a major knowledge gap in a fundamental aspect of aerobic metabolism. The objectives of this proposal are to reveal how the flux of heme through the heme a biosynthetic pathway is coupled to CcO biosynthesis and the key protein-protein interactions that assist and help regulate these processes. At least three different proteins are critical for the biosynthesis of heme A and its subsequent insertion into subunit 1 (Cox1) of CcO: heme O synthase (HOS), heme A synthase (HAS) and Surf1/Shy1. We hypothesize that HOS and HAS form distinct, large protein complexes that alter enzymatic activity. In our model, the HAS complex delivers heme A to Cox1 while Surf1/Shy1 aids in this process by stabilizing the newly inserted heme A and preventing its loss during assembly. We further hypothesize that the activity of HOS and HAS is coupled to Cox1 translation. These hypotheses will be tested by pursuing two specific aims: 1) characterize the relationship between heme A biosynthesis and CcO assembly, and 2) ascertain the significance of the HOS and HAS protein complexes. To accomplish these goals, we will employ both in vivo and in vitro studies using Rhodobacter sphaeroides and Saccharomyces cerevisiae as models. The proposed research is significant because it addresses two of the most central but also least understood aspects of CcO biogenesis. (1) How does the cell regulate the production of the vital yet toxic heme a cofactor? (2) How is heme A inserted into CcO? Our approach is innovative because we are simultaneously employing both R. sphaeroides and S. cerevisiae to capitalize on the stability and relative simplicity of bacterial oxidases while validating our results in a genetically tractable eukaryote. The synergy provided by employing both systems positions us to make rapid progress addressing these essential questions of CcO biogenesis. PUBLIC HEALTH RELEVANCE: Heme A is an obligatory cofactor in cytochrome c oxidase, the enzyme responsible for converting O2 to water and storing the energy as an electrochemical gradient during respiration. The proposed research on heme A homeostasis is relevant to public health because improper regulation of heme a levels leads to a variety of mitochondrial diseases including anemia, Leigh Syndrome, and infantile cardiomyopathy. We will reveal how heme a biosynthesis is regulated and how this critical cofactor is inserted during cytochrome c oxidase biogenesis.